Friedreich's ataxia (FA), a neurodegenerative disease, is the most common inherited autosomal recessive ataxia, and is caused in 98% of all cases by expansion of GAA repeats in the first intron of the frataxin gene (FXN) (1). In healthy individuals the alleles may contain up to 40 GAA repeats, whereas expanded alleles in FA patients can consist of 90 to 1700 repeats (2). The GAA repeat expansion leads to major reduction in frataxin mRNA and low levels of the protein in mitochondria (2). Also carriers (heterozygous for the expanded allele) show ˜50% reduction of mRNA and protein levels compared to normal expression, although they do not show any symptoms (1). Frataxin deficiency causes excessive free radical production, dysfunction of Fe—S center containing enzymes, and progressive iron accumulation in mitochondria (3).
Friedreich's ataxia is a deadly disease and affects people at an early age. Today, there is not any way to cure or prevent the disease and current therapy can only treat the symptoms. The number of expanded GAA repeats in FA is directly correlated to the age-of-onset and severity of the disease.
Expanded GAA repeats form an intramolecular triple-helix (triplex), so-called H-DNA, (FIG. 1) in supercoiled plasmids isolated from E. coli (4). Several models representing the triplex structures formed at expanded GAA repeats are proposed, and direct evidence for a pyrimidine motif H-DNA structure at pathological GAA expansions in vitro has recently been provided (5). Also, formation of a higher order structure named “sticky DNA” has been observed in frataxin GAA repeats-containing plasmids using gel electrophoresis and atomic force microscopy (4). The molecular structure of sticky DNA is not resolved; however, current evidence demonstrates that sticky DNA forms as one long intramolecular triplex structure or by the association of two triplexes.
Structural properties of GAA repeats may affect the stability of the repeat length as well as expression of frataxin (6). Long GAA repeats were shown to stall replication in vivo in Saccharymyces cerevisiae (7) and inhibit transcription in vitro and in transfected cells (8). The observed effects on DNA replication and transcription are dependent on the length and orientation of the GAA repeats in plasmids, which correlate with formation of the specific DNA structure (H-DNA). Finally, the GAA repeats are associated with a pattern of DNA methylation and histone acetylation in the adjacent regions and the formation of silenced chromatin. The presence of H-DNA and higher order structures within the GAA repeats is believed to recruit chromatin-remodeling protein complexes that maintain a close chromatin structure leading to down-regulation of frataxin gene transcription.
Numerous data have demonstrated that analysis of GAA repeats constitute an essential part in the diagnosis of FA along with clinical diagnosis. Molecular genetic tests are also performed to identify carriers and in prenatal testing. Current FA diagnostic methods involve polymerase chain reaction (PCR) analysis and Southern blotting technique. The PCR test is performed by amplification of the GAA repeat-containing DNA region in the frataxin gene. The different PCR reactions that have been employed to map GAA repeat expansions are classical PCR, long-range PCR or triplet-primed PCR (TP-PCR). In all cases, the size of the PCR fragment is analyzed using agarose-gel electrophoresis and DNA sequencing. In most cases, both PCR and Southern blot are combined to complement the results.
Problems encountered during amplification of medium and long sized GAA repeats (number of repeats >200) using PCR have been reported. The repetitive nature of the expanded sequence and its ability to adopt H-DNA and higher order DNA structures are the two main factors causing polymerase pausing leading to false results.
Thus, there is still a need in the art for alternative or improved methods for detecting the expanded GAA repeats to be used in the diagnosis of Friedreich's ataxia. Also, there is a need for therapies in order to treat and/prevent Friedreich's ataxia.